Project Summary Coronary artery heart disease (CAD) is the leading cause of death in the United States. Although pre-ischemic preconditioning (IPC) has been accepted as the gold standard method of cardioprotection against coronary ischemic injury; its application remains difficult in the clinical setting because of the requirement of triggering episodes of coronary ischemia reperfusion. Thus, understanding the molecular adaptations induced by IPC is of paramount importance and will help developing novel therapy. Recently, we found that the valosin- containing protein (VCP), an ATPase-associated protein, previously uncharacterized in the heart, is a key mediator of IPC-induced cardiac protection. Specifically, we found that VCP is upregulated in the IPC hearts, and that overexpression of VCP protects cardiomyocytes from the stress-induced apoptosis in vitro and dramatically reduces the infarct size of coronary ischemic injury in vivo, as effective as those conferred by the IPC. Additionally, we found that VCP expression in cardiomyocytes leads to accumulation and activation of the inducible nitric oxide synthase (iNOS) in mitochondria and S-Nitrosylation (SNO) of mitochondrial proteins. We also found that VCP expression leads to activation of the target of rapamycin complex 2 (mTORC2) and mTORC2-dependent signaling. Furthermore, we have identified a regulatory N-domain of VCP that is essential for VCP-mediated cyto-protection. Collectively, our data may have discovered a previously unrecognized role of VCP in cardiac protection and suggests that VCP is a potential novel candidate for therapy of CAD. However, despite these exciting preliminary findings, the physiological significance of VCP in the protection of coronary ischemic injury has not been exquisitely established, and the underlying molecular mechanisms by which VCP mediates mTOCR2 activation and by which VCP/mTORC2 signaling leads to iNOS mitochondrial translocation and function have been largely unknown. Our central hypothesis is that VCP is a potent mediator of cardioprotection against coronary ischemic injury; it promotes cardiac survival by activating mTORC2 and mTORC2-mediated iNOS mitochondrial translocation, leading to preservation of mitochondrial function and enhancement of myocardial tolerance to coronary ischemic injury, We will test our central hypothesis by a comprehensive set of experiments under two specific aims. Under Aim 1 we will define the physiological significance and therapeutic potential of VCP in coronary ischemic injury in mice. Under Aim 2, we will determine the molecular mechanisms by which VCP promotes mTORC2 activation and iNOS mitochondrial translocation and cardiomyocyte survival. We expect that this study will advance the field by establishing the protective role of VCP in coronary ischemic injury and opening new avenues into research for better therapy of CAD.